Ординатура / Офтальмология / Английские материалы / Anti-VEGF_Bandello, Parodi, Augustin_2010

therapy in ocular diseases. After an outline of the treatment principles, it covers a large number of topics, including age-related macular degeneration, pathologic myopia, angioid streaks, inflammatory diseases, hereditary dystrophies, retinal vein occlusions, diabetic retinopathy, ocular tumors, and anterior segment neovascularizations. We hope that each chapter will stimulate the interest of readers working in this field.

Francesco Bandello, Milan

choriocapillary layer. The physiological stability of the ocular vascular system is an equilibrium between angiostatic and angiogenic factors. The vasculature is stable as long as the angiostatic factors are ahead. Pathologic conditions such as ischemia or inflammation shift the balance towards angiogenic factors which are released by the damaged cells. On the other hand the unpredictable appearance of neovascularization during dry AMD which cannot be prevented by anti-inflammatory treatment strongly points out that also a loss of angiostatic factors alone can lead to instability of the constructive vascular boundaries of the eye.

The strong angiostasis that is crucial for the function of the eye is maintained by angiostatic factors in every involved tissue starting from the specialized guards of the blood-retinal barrier down to unspecific ingredients of the blood fluid. The angiostatic effect is not only locally distributed but also stepwise during stages of angiogenesis. Due to the defensive nature of static concepts, not only active components such as inhibitor proteins but also passive stabilizing members of the extracellular matrix can be accounted to the angiostatic system.

Thus, collagens, elastins and fibrin constitute a first barrier for angiogenesis. These molecules have to be actively degraded and the respective proteases are controlled by protease inhibitors. Tissue inhibitors of metalloproteinases are specific metalloproteinase inhibitors while the serum component α2-macroglobulin unspecifically inhibits metalloproteinases. Another protein that interferes with pericellular proteolysis required for migration and proliferation of endothelial cells is thrombospondin which is present in platelet granules and is released following platelet activation. If proteolytic degradation of capillary basement membranes occurs, a fragment of the collagen type 18 called endostatin is released. It specifically inhibits proliferation of endothelial cells and angiogenesis.

Other passive components of vascular stability are the VE cadherins that are involved in intercellular tight junctions – the constituting basis of the blood-retinal barrier. VE cadherins are members of a large family of adhesion proteins called cadherins that build intercellular contacts like desmosomes throughout the body. VE cadherins have to be degraded before angiogenesis can occur. Their degradation is triggered by vascular endothelial growth factors (VEGF) via the VEGFR-2 receptor.

More active components of vascular structural stability of the eye are proteins that are secreted by the cells of the blood-retinal barrier. A protein that maintains stability after maturation of newly grown capillaries is angiopoietin-1. It is produced by pericytes. Its presence in mature capillaries improves continuity of the basal membranes and the adherence of pericytes to endothelial cells. During angiogenesis it promotes capillary growth. It is antagonized by angiopoietin-2 which binds to the same endothelial cell-specific receptor Tie-2. TGF-β has among its many other effects a similar role as it is secreted by pericytes and stabilizes the basal membrane of newly built capillaries.

Pigment epithelium-derived factor is a cytokine that despite its name is produced in many human cells including endothelial cells and retinal pigment epithelial cells

where it was originally detected. Among other effects it is a potent inhibitor of angiogenesis. It also has immunomodulatory features and contributes by this indirectly to prevention of neovascularization.

The vasoinhibins act as negative feedback regulators upon the effect of VEGF. They are upregulated in endothelial cells by VEGF and specifically inhibit migration and proliferation of these. Angiostatin also specifically inhibits proliferation of endothelial cells. It is a fragment of plasminogen and therefore exists as a plasma factor throughout the body.

Angiogenic Factors

The growth of new blood vessels is an important natural process occurring in the body, both in health and disease. Angiogenesis is a physiological process involving the growth of new blood vessels from preexisting vessels whereas vasculogenesis describes the formation of vascular structures from circulating or tissue-resident endothelial stem cells (angioblasts) which proliferate into de novo endothelial cells.

The healthy body controls angiogenesis through a series of ‘on’ and ‘off’ switches. The main ‘on’ switches are known as angiogenesis-stimulation growth factors, or simply angiogenic factors. Stimulation of angiogenesis is performed by various angiogenic proteins, including several growth factors, whereas the VEGF family has been demonstrated to be a major contributor to angiogenesis. Additionally, a large number of mediators exist which are involved in angiogenesis like insulin-like growth factor, the family of fibroblast growth factor, interleukins, angiopoietins, epidermal growth factor, transforming growth factors, platelet-derived growth factor, tumor necrosis factor-α and vascular endothelial cadherin.

The balance between angiogenesis and inhibitors of new vessel growth is controlled by a sophisticated interaction between different factors and mediators which will be described explicitly in the following chapter.

Prof. A.J. Augustin Augenklinik Moltkestrasse 90

DE–76133 Karlsruhe (Germany)

Tel. +49 721 9742001, Fax +49 721 9742009, E-Mail albertjaugustin@googlemail.com

quiescent. The growth potential of smaller blood vessels, however, is retained and is employed during wound healing and tissue regeneration.

Beyond its physiological roles, angiogenesis is also a hallmark of many pathological conditions, including neovascular diseases in the eye [3–5]. Excessive angiogenesis occurs when diseased cells produce abnormal amounts of angiogenic factors, overwhelming the effects of natural angiogenesis inhibitors. As the newly formed vessels mainly serve a role in a wound healing response, they usually do not restore the tissue integrity, but rather cause visual impairment when they are located in normally avascular, transparent tissues such as the cornea and vitreous. Strategies for inhibition of angiogenesis include approaches that can block the angiogenesis cascade at several steps [4, 6].

Angiogenesis: Mechanisms and Molecular Mediators

Endothelial Cell Differentiation

All blood vessels are lined by endothelial cells (ECs), which form the interface between circulating blood in the lumen and the rest of the vessel wall. Under normal conditions, ECs are a remarkably quiescent cell type, undergoing division approximately once every 1,000 days, but when activated, cell division can occur every 1–2 days [7]. Sprouting angiogenesis requires selection of ECs from an existing blood vessel which will be activated to form the new vessel, while at the same time, surrounding ECs remain quiescent in their current position. From recent studies a model has emerged in which ECs differentiate into three specialized cell types with distinct phenotypes during angiogenesis (fig. 1) [8–10]. First, a single ‘tip cell’ develops. This EC breaks down the basal lamina, emerges from its parent blood vessel and becomes the leading cell of the sprouting vessel. The tip cell migrates into the extracellular matrix and senses microenvironmental attractive and repulsive signals for guidance. Secondly, following directly behind the migrating tip cell, other ECs differentiate under the influence of the adjacent tip cell into ‘stalk cells’ that proliferate and bridge the gap between the tip cell and the parent vasculature. Stalk cells generate the blood vessel lumen through the formation of intracellular vacuoles, a process called ‘lumenogenesis’. Thirdly, ECs behind the stalk cells differentiate into ‘phalanx cells’, and align in a smooth cobblestone monolayer, becoming the most inner cell layer in the new blood vessel. Phalanx cells no longer proliferate, express tight junctions and make contact with mural cells.

Angiogenesis Inducers and Inhibitors

Angiogenesis is tightly controlled by closely interacting angiogenic and angiostatic factors, and their balance ultimately determines if, where and when the ‘angiogenic switch’ is turned on with angiogenesis as the result [2, 9]. Over the past decades, numerous inducers of angiogenesis have been identified, including the members of

Tip cell

Breaks down basal lamina

Emerges from its preexisting vessel

Migrates through extracellular matrix

Extends filopodia to explore for guidance clues

Controls tip and stalk cell numbers

Stalk cells

Form the stalk of the vessel sprout

Proliferate

Form vacuoles to generate vessel lumen

Lay down extracellular matrix

Phalanx cells

Become quiescent

Align and form a smooth monolayer

Express tight junctions

Make contact with mural cells (pericytes)

Fig. 1. Representative model of sprouting angiogenesis. At least three different angiogenic specialized endothelial cells (white) are required, each with a distinct cellular fate. In addition, the new blood vessel becomes surrounded by pericytes (dark gray) and a new basal lamina (light gray).

the vascular endothelial growth factor (VEGF) family, angiopoietins, transforming growth factors (TGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), tumor necrosis factor-α (TNF-α), insulin-like growth factor (IGF), vascular endothelial-cadherin (VE-cadherin), interleukins and the members of the fibroblast growth factor (FGF) family. In addition, there is a plethora of growth factors, hormones and metabolites that have been reported to directly or indirectly stimulate physiological and pathological angiogenesis (table 1) [11, 12]. Not all of these factors are specific for ECs. Consistent with a major role for hypoxia in the overall process of angiogenesis, a large number of angiogenic factors involved in various stages of angiogenesis are independently responsive to hypoxia [13]. The VEGF family of proteins is the most important family of angiogenic factors that controls blood vessel formation.

Endogenous inhibitors of angiogenesis are defined as proteins or fragments of proteins that can inhibit the formation of blood vessels [14]. Angiogenesis inhibitors

Table 1. Major angiogenic factors

Table 1. Continued

EC = Endothelial cell, PC = pericyte, ECM = extracellular matrix, BL = basal lamina.

can be detected in circulating blood, suggesting that they function in the angiogenic switch as endogenous angiostatic regulators under physiological conditions. Various inhibitors of angiogenesis have been found in the body, including thrombospondin, angiostatin, endostatin and pigment epithelium-derived factor (PEDF) (table 2) [12, 14].

The VEGF Family and Their Receptors

In mammals, the VEGF family includes VEGF-A (also referred to in this review as VEGF), VEGF-B, placenta growth factor (PlGF), VEGF-C, VEGF-D, and the viral VEGF homologue VEGF-E. VEGFs bind selectively with different affinities to at least five distinct receptors: VEGF receptor-1 (VEGFR-1), also called Flt-1; VEGFR-2, also called Flk-1; VEGFR-3, also called Flt-4; neuropilin-1 (NRP-1), and NRP-2 [5, 15, 16]. The VEGFRs are members of the tyrosine-kinase receptor superfamily. Ligand binding to the extracellular immunoglobulin-like domain induces receptor dimerization. VEGFR-2 is considered to be the major receptor responsible for mediating the

Table 2. Major endogenous angiostatic factors

EC = Endothelial cell, PC = pericyte, ECM = extracellular matrix, BL = basal lamina.